Amyloid fibril structure from the vascular variant of systemic AA amyloidosis

Systemic AA amyloidosis is a debilitating protein misfolding disease in humans and animals. In humans, it occurs in two variants that are called ‘vascular’ and ‘glomerular’, depending on the main amyloid deposition site in the kidneys. Using cryo electron microscopy, we here show the amyloid fibril structure underlying the vascular disease variant. Fibrils purified from the tissue of such patients are mainly left-hand twisted and contain two non-equal stacks of fibril proteins. They contrast in these properties to the fibrils from the glomerular disease variant which are right-hand twisted and consist of two structurally equal stacks of fibril proteins. Our data demonstrate that the different disease variants in systemic AA amyloidosis are associated with different fibril morphologies.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. The patient details are presented as a cohort, no direct identifiers are presented in the paper.
Two patients with vascular variant of AA amyloidosis (1 male and 1 female). Tissue from up to four patients with glomerular variant of AA amyloidosis (3 males and 1 female) was used to illustrate known features of the previously described disease variant.
The patients were selected on the pattern of renal AA amyloid deposition -vascular or glomerular deposition. The patients were not selected biased on gender, sex or any other relevant parameters that influences the study.
Tissue materials were collected at the University Medical Center Groningen after obtaining informed consent from the patients or their relatives, who did not receive any compensation. All relevant regulations and legal requirements, including ethical approval from relevant authorities at Groningen University, were observed during material collection. The biochemical work at Ulm University was conducted based on a permission from the Ulm University Ethics Commission (203/18).
No established methods or statistical calculations were considered for the sample size. The samples size is dependent on the availability of the tissue. The glomerular AA amyloidosis is more common and has four cases, while the vascular AA amyloidosis is rarer and has two cases.
Vascular AA patient I: 77,061 helical segments were retained from initially 168,956 helical segments. Vascular AA patient II: 52,098 helical segments were retained from initially 96,599 helical segments.
The reconstructed density map confirmed by two consecutive dataset from two different patients. The reproducibility of other experimental results relies on atleast three independent replicated experiments, unless stated otherwise.
Not relevant to the study as it is a two case study from one cohort.
The analysis is a two case study that does not report statistical comparisons between different cohorts.

Introduction
Amyloidosis is a group of diseases that have in common the extracellular deposition of fibrillar proteins with a specific biochemical conformation known as ß-pleated sheets. Approximately 20 different precursor proteins that may be deposited as amyloid fibrils have been identified (3). Amyloid proteins deposited in the tissues can be identified by Congo Red staining, and chemically classified via amino acid sequence studies, by immunochemistry, or via immunocytochemistry (1). Amyloid A (AA) is an extracellular deposited insoluble fibrillar protein, highly resistant to proteolytic degradation, and produced from the precursor protein, serum amyloid A (SAA) (3). Before the therapy of a suspected amyloid disease can be planned, both the presence of amyloid and its chemical origin must be known (2). Reagent provided Monoclonal mouse antibody provided in liquid form as cell culture supernatant dialysed against 0.05 mol/L Tris/HCL, pH 7.2, and containing 15 mmol/L NaN3. Clone: mc1 (4, 5). Isotype: IgG2a, kappa. Mouse IgG concentration: see label on vial. Immunogen An equal mixture of human amyloid A coupled to horseradish peroxidase and human amyloid A coupled to high molecular weight kininogen (5).

Specificity
In micro-ELISA, the antibody reacts with amyloid A and the serum precursor of amyloid A indicating cross-reactivity among amyloid A protein and the serum precursor of amyloid A. In contrast, no reactivity to other non-AA amyloid fibril proteins or human serum proteins, such as albumin, transferrin, and IgG is seen (4, 5). In immunocytochemistry, the antibody labels tissues from AA patients, but shows no reactivity with a host of unknown antigens in tissue sections of various organs (5), nor with amyloid types Akappa, Alambda and amyloid fibril proteins in familial amyloid polyneuropathy and senile cardiovascular amyloidosis, respectively. Neither are Alzheimer's amyloid plaques, amyloid in microangiopathy, lichen amyloidosis, and senile islets of Langerhans labelled by the antibody (5-7). Precautions 1. For professional users. 2. This product contains sodium azide (NaN3), a chemical highly toxic in pure form. At product concentrations, though not classified as hazardous, sodium azide may react with lead and copper plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush with large volumes of water to prevent metal azide build-up in plumbing. 3. As with any product derived from biological sources, proper handling procedures should be used.

Storage
Store at 2-8 degree C. Do not use after expiration date stamped on vial. If reagents are stored under any conditions other than those specified, the user must verify the conditions. There are no obvious signs to indicate instability of this product. Therefore, positive and negative controls should be run simultaneously with patient specimens. If unexpected staining is observed which cannot be explained by variations in laboratory procedures and a problem with the antibody is suspected, contact our Technical Services.
Specimen preparation Paraffin sections: The antibody can be used for labelling paraffin-embedded tissue sections fixed in formalin. Pre-treatment of tissues with proteinase K or heat-induced epitope retrieval is recommended. For heat-induced epitope retrieval, optimal results are obtained with Dako Target Retrieval Solution, code No. S 1700, Dako Target Retrieval Solution, High pH, code No. S 3308, 10 mmol/L citrate buffer, pH 6.0, or 10 mmol/L Tris buffer, 1 mmol/L EDTA, pH 9.0. The tissue sections should not dry out during the treatment or during the following immunocytochemical staining procedure. Frozen sections and cell preparations: The antibody can be used for labelling frozen sections (5).
Staining procedure Dilution: Monoclonal Mouse Anti-Human Amyloid A, code No. M 0759, may be used at a dilution range of 1:50-1:100 when applied on formalin-fixed, paraffin-embedded sections of human kidney from a patient with amyloidosis A and using 20 minutes heatinduced epitope retrieval in Dako Target Retrieval Solution, code No. S 1700, and 30 minutes incubation at room temperature with the primary antibody. Optimal conditions may vary depending on specimen and preparation method, and should be determined by each individual laboratory. The recommended negative control is Dako Mouse IgG2a, code No. X 0943, diluted to the same mouse IgG concentration as the primary antibody. Unless the stability of the diluted antibody and negative control has been established in the actual staining procedure, it is recommended to dilute these reagents immediately before use, or dilute in Dako Antibody Diluent, code No. S 0809. Positive and negative controls should be run simultaneously with patient specimen. Visualization: DAKO LSAB™+/HRP kit, code No. K 0679, and DAKO EnVision™+/HRP kits, code Nos. K 4004 and K 4006, are